Epigenetic inactivation of microRNA in male germ cell cancer Personnel: Lee, Cheung, Chan, Rennert MicroRNAs (miRNAs) are a class of small non-coding RNAs that have been shown to be deregulated in many diseases including cancer. An intertwined connection between epigenetics and miRNAs has been supported by the recent identification of a specific subgroup of miRNAs called epi-miRNAs that can directly and indirectly modulate the activity of the epigenetic machinery. Using a genome-wide approach for studying differential methylation in testicular germ cell tumor cell line, we previously identified a novel hypermethylated locus on chromosome 1. Genomic mapping revealed that the hypermethylated region overlapped with a mircroRNA candidate, miR-199a and its upstream promoter region. Bisulfite sequencing and treatment with DNA methyltransferase inhibitor 5-aza in the Ntera-2 testis cancer cells confirmed the same observation in the tiling microarray data. Changes in DNA methylation are a hallmark of cancer, and are frequently associated with cancer progression. Epigenomic profiling revealed a conserved region in intron-14 of dynamin 3, located at 1q24.3; its hypermethylation correlated with testicular cancer progression, and silencing of miR-199a. Re-expression of miR-199a in testicular cancer cells suppressed cell growth, cancer migration, invasion, and metastasis. miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with cancer progression. We identified an embryonal carcinoma antigen, podocalyxin-like protein 1 (PODXL), as a target of miR-199a-5p. PODXL is an anti-adhesive protein overexpressed in aggressive testicular cancer. Knockdown of PODXL suppressed cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests that PODXL is one of the downstream effectors mediating cancer invasion and metastasis. This report links DNA methylation, miR-199a dysregulation, and PODXL expression as a mechanism to explain testicular cancer progression.